The Orthopoxvirus genus of the family Poxviridae includes both the causative agent of smallpox, variola virus, and the vaccine used in its eradication, vaccinia virus. Recent events have renewed interest in Orthopoxviruses because of concerns that variola virus or one of its close relatives could be used as a biological weapon. Orthopoxviruses produce both enveloped and unenveloped virions and remarkably both types are infectious. While non-enveloped virions make up the majority of progeny virions the enveloped form (EV) is required for cell-to-cell spread, systemic infection and virulence. The major objective of work is to determine the molecular mechanism employed by envelope virus specific proteins to coordinate the intracellular envelopment of Orthopoxviruses. Our hypothesis is that the lumenal domain of three EV specific proteins, A33, A34 and B5, interact and that interaction is required for exit from the ER and subsequently proper sub-cellular targeting of B5, which is the main target of neutralizing antibodies, to the site of intracellular envelopment and subsequently into infectious enveloped virions. The specific aims are: 1) Mapping residues of A33, A34 and B5 required for proper targeting and interaction. The studies proposed here will use an innovative live trans complementation microscopy assay and co-IP to map regions of interaction. 2) Characterization of the relationship between A33, A34 and B5 in EV production. Recombinant viruses will be created that express A3R, A3R and BR mutations defined in Aim1 to characterize the role the interaction has during viral morphogenesis. 3) Temporal and spatial characterization of A33, A34 and B5 interaction. The innovative fluorescent assays FRET and BiFC will be used to track the interaction in live cells. Mapping sites of interaction on these proteins will help define functional domains and provide for a better understanding of their structure and function. The B5R protein is required for the formation of enveloped poxviruses. We will investigate how this important protein is incorporated into and coordinates the formation of newly made viruses. A better understanding of this process will provide new targets for antivirals directed against poxviruses. [unreadable] [unreadable] [unreadable]